As spermatozoa progress along the epididymal duct, they are bathed in a specialized luminal fluid microenvironment which is crucial for their maturation and survival. As the spermatozoa mature, they need constant protection against oxidative damage and from harmful effects of drugs and toxic chemicals. Damaged spermatozoa may lead to abnormal offspring. In many tissues such as the lung and kidney, glutathione (glu-cys-gly) has been shown to play an important role in antioxidant defense, and it seems likely that it plays a similar role in the epididymis. The focus of this proposal is to determine the manner by which luminal glutathione is regulated by the epididymal epithelium. We will use in vivo micropuncture and microperfusion techniques to determine the origin of glutathione in the luminal fluid of different epididymal regions. We will determine the activity of gamma-glutamyl transpeptidase (GGT), the only known enzyme that can cleave glutathione to yield glutamate and the dipeptide cysteine- glycine. Experiments will also be performed to determine the catalytic activity of dipeptidase which cleaves cysteine-glycine into its constituent amino acids. The hypothesis that GGT can transport cysteine and glycine from the lumen into the epididymal cells will be tested. We have previously found that luminal GGT catalytic activity declines from caput to cauda. Therefore, experiments will be designed to determine the mechanism(s) of the loss of GGT activity within the cauda epididymal region, whether it is due to degradation of the enzyme by proteases and/or endoglycosidases located within the luminal fluid and/or on the apical surface of cauda epithelial cells. Using molecular biological techniques we will identify the epididymal cell type(s) that express GGT mRNA. Hence, we will identify those cells that play an important role in the regulation of epididymal glutathione. It is critical that we understand the mechanisms by which the epididymis maintains an environment that will protect spermatozoa from oxidation and from harmful effects of various drugs and xenobiotics; oxidative defense is crucial for the survival of spermatozoa. This proposal is part of a long term goal to understand the mechanisms by which the epididymis maintains an optimal luminal microenvironment for sperm maturation and survival. The findings from this proposal will also provide fundamental information for the development of a male contraceptive and treatment of certain forms of male infertility.